Download Paclitaxel- and lapatinib-loaded lipopolymer micelles overcome multidrug resistance in prostate cancer.

Drug Deliv. and Transl. Res. (2011) 1:420–428
DOI 10.1007/s13346-011-0042-2
RESEARCH ARTICLE
Paclitaxel- and lapatinib-loaded lipopolymer micelles
overcome multidrug resistance in prostate cancer
Feng Li & Michael Danquah & Saurabh Singh & Hao Wu & Ram I. Mahato
Published online: 25 October 2011
# Controlled Release Society 2011
Abstract Paclitaxel is a potent chemotherapeutic agent for
treating refractory prostate cancer. However, its prolonged
treatment develops multidrug resistance. Since lapatinib
interacts with and inhibits P-gp activity, our objective was
to determine whether the combination therapy of these two
drugs can synergistically treat resistant prostate cancer. Our
recently synthesized lipopolymer, poly(ethylene glycol)block-poly(2-methyl-2-carboxylpropylene carbonate-graftdodecanol) (PEG–PCD), was used to efficiently load both
drugs into PEG–PCD micelles since they are hydrophobic.
Lapatinib inhibited P-gp function but not its expression.
Co-treatment of DU145-TXR cells with 0.5 μM paclitaxel
and 5 μM lapatinib resulted in up to 138-fold reversal
compared to paclitaxel alone. These formulations killed
almost 70% and 80% of DU145-TXR cells when 0.5 μM
paclitaxel was combined with lapatinib at a dose of 1 and
5 μM, respectively, while monotherapy had no effect.
Combination therapy induced apoptosis and cell cycle
arrest at mitotic phase. Xenograft tumor growth in athymic
nude mice was significantly regressed when PEG–PCD
micelles carrying lapatinib and paclitaxel were given
intravenously twice a week. Furthermore, this combination
therapy synergistically decreased antiangiogenic activity
compared to the control or their monotherapy. In conclusion, lipopolymeric micelles carrying lapatinib and paclitaxel have the potential to treat resistant prostate cancer and
can successfully deliver drugs to tumors while minimizing
toxic effects associated with solubilizing agents.
F. Li : M. Danquah : S. Singh : H. Wu : R. I. Mahato (*)
Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center,
19 S Manassas, CRB 224,
Memphis, TN 38103-3308, USA
e-mail: [email protected]
URL: http://www.uthsc.edu/pharmacy/rmahato
Keywords Lipopolymer . Prostate cancer . Lapatinib .
Paclitaxel . Multidrug resistance
Introduction
Prostate cancer is the second common male cancer in the
USA. Although the majority of patients respond well to
androgen ablation therapy, chemotherapy, and radiotherapy
at the beginning, many patients relapse over time and
become resistant to chemotherapy [1, 2]. This is mainly due
to the over-expression of multiple drug-resistant (MDR)
transporters in prostate cancer cells (Fig. 1). These transporters, such as P-glycoprotein (P-gp), breast cancer
resistance protein, and multiple drug resistance protein,
increase drug efflux and reduce drug accumulation in tumor
cells [3–5]. The prognosis of patients with MDR cancer is
poor, due to the lack of effective clinical interventions.
Also, many commonly used chemotherapy drugs such as
paclitaxel have inherent toxicity associated with their use.
Thus, there is an urgent need for effective therapies for
MDR prostate cancers while reducing the side effects
associated with the drug or its delivery mechanism. Several
strategies have been used to overcome MDR, including
developing of novel anticancer agents [6] using novel
delivery systems, such as liposomes [7], solid lipid nanoparticles [8], polymer–drug conjugates [9], and the use of
MDR transporter inhibitor [10].
Lapatinib is a dual tyrosine kinase inhibitor what targets
both human epidermal growth factor receptor (EGFR) and
epidermal growth factor receptor 2 (HER2). Lapatinib
blocks autophosphorylation reaction and the downstream
of events. Lapatinib was approved by the FDA for treating
advanced breast cancers [11]. Studies indicate that lapatinib
can inhibit the function of ATP-binding cassette (ABC)
Drug Deliv. and Transl. Res. (2011) 1:420–428
421
Lapatinib
Resistance
MDR transporters
Sensitization
Paclitaxel
Fig. 1 Schematic diagram showing paclitaxel and lapatinib combination therapy for treating MDR cancer
transporters and thus sensitize MDR cancer cells to
chemotherapeutic agents (Fig. 1) [12–14]. Lapatinib has
also been reported to decrease the percentage of cancer
stem cells and improved the long-term survival of patients
[15]. Moreover, several studies have demonstrated the
potential of lapatinib for treating prostate cancer. For
example, Liu et al. have shown that inhibiting HER2 using
lapatinib is capable of disrupting androgen function in
prostate cancer [16]. Also, Shaw and coworkers elegantly
demonstrated targeting of Hedgehog-ErbB signaling using
cyclopamine and gefitinib or lapatinib inhibited proliferation of androgen-independent prostate cancer cells [17].
Additionally, it is noteworthy that lapatinib is currently
being evaluated in phase II clinical trial for the treatment of
early stage hormone-dependent or metastatic prostate
cancer [18]. Phase III clinical trials using combination of
lapatinib and paclitaxel have significantly improved clinical
outcomes in HER-2-positive breast cancer patients [19].
This addition of lapatinib allows the use of a lower dose of
chemotherapeutic drug which can reduces the toxic side
effects of therapy.
Polymeric micelles are prepared from amphiphilic
diblock copolymers and used for hydrophobic drug
delivery. It forms a core–shell structure by self-assembly.
The hydrophobic core of micelles is capable of loading
hydrophobic drugs and thus avoids the use of toxic
solubilizing agents in the delivery of poorly soluble
anticancer drugs [20, 21]. The stealth properties associated
with hydrophilic PEG corona of micelles prevent their
aggregation, restrict plasma protein adsorption, prevent
recognition by the reticuloendothelial system, and minimize
rapid elimination from the bloodstream. The small size of
micelles ensures their accumulation preferentially in the
tumor via effective permeation and retention effects [22,
23]. For targeted delivery of drugs into tumors, the micelles
need to be stable in the blood circulation. The stability of
micelles can be improved through the engineering of the
hydrophobic core. In our previous study, we designed a
lipopolymer poly(ethylene glycol)-block-poly(2-methyl-2carboxylpropylene carbonate-graft-dodecanol) (PEG–PCD)
for micellar drug delivery. This lipopolymer showed
significantly improved stability and good drug loading for
many poorly soluble drugs [5]. In the current study, we
used lipopolymer PEG–PCD micelles to formulate lapatinib
and paclitaxel. Firstly, we optimized the micelle formulation and characterized the formulation with analytical and
biophysical methods. Then, the anticancer effects of
lapatinib- and paclitaxel-loaded micelles were determined
with in vitro cell-based assays, focusing on their effects to
overcome MDR in prostate cancers. We also tested their
abilities to inhibit MDR tumor growth in vivo with athymic
mice xenograft tumor models established with MDR
prostate cancer cells. Our aim in this study is to replace
the use of commonly used solubilizing agents such as
DMSO and Cremaphor-EL which are known to induce
cellular and organ toxicity.
Materials and methods
Materials
Hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, dodecanol, triethylamine, 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU), 2,2-bis(hydroxymethyl) propionic acid, methoxypoly(ethylene glycol) (mPEG; Mn =
5,000, PDI=1.03) and all other reagents were purchased
from Sigma Aldrich (St. Louis, MO, USA) and used as
received. DU145-TXR cells were provided by Professor
Evan T. Keller of the University of Michigan.
Synthesis of poly(ethylene glycol)-block-poly(2-methyl-2carboxyl-propylene carbonate-graft-dodecanol)
PEG–PCD was synthesized as described previously
[24]. Firstly, poly(ethylene glycol)-block-poly(2-methyl2-benzoxycarbonyl-propylene carbonate) (PEG–PBC)
was synthesized through a ring-opening polymerization
from monomer MBC, using mPEG as a macro-imitator
and DBU as a catalyst. Then, protective benzyl groups in
PEG–PBC was removed by hydrogenation to get poly
(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene
carbonate) (PEG–PCC). Finally, lipopolymer PEG–PCD was
obtained after conjugation of dodecanol lipid pendant groups
to PEG–PCC.
Lipopolymers and intermediate polymers were characterized with following methods: (1) nuclear magnetic
resonance (NMR). 1H NMR spectra were recorded on
Varian (500 MHz) using deuterated chloroform (CDCl3) as
a solvent unless otherwise noted. The chemical shifts were
422
calibrated using tetramethylsilane as an internal reference
and given in parts per million; (2) gel permeation
chromatography (GPC). The weight (Mw) and number
(Mn) average molecular weight and polydispersity index
of synthesized polymers were determined by a Waters GPC
system equipped with a GPC column (AM Gel 103/5) and a
differential refractive index detector. THF was used as an
eluent at a flow rate of 1 mL/min. A series of narrow
polystyrene standards (700–40,000 g/mol) were used for
calibration.
Preparation and characterization of lipopolymeric micelles
Lipopolymer micelles were prepared with a film dispersion
method as previously described with some modifications
[6]. Briefly, 30 mg of lipopolymers and given amount of
drugs were dissolved in 0.5 mL CH2Cl2, before the solvent
was removed under reduced pressure. The resulting film
was hydrated in 3 mL saline (0.9%) and sonicated for
1 min. The residual free drug was removed by centrifugation at 12,000 rpm for 5 min. The supernatant was filtered
using a 0.22-μM filter. Whenever we used both paclitaxel
and lapatinib, they were co-dissolved for drug loading in
the same micelles.
The particle size distribution of micelles was determined
by dynamic light scattering with Malvern Nano ZS. To
determine drug loading, 10 μL of drug-loaded micelle
solution was dissolved with acetonitrile and the drug
concentration determined using a reverse-phase high performance liquid chromatography (RP-HPLC; Waters, Milford,
MA, USA) with a UV detector at 227 nm. A C18 column
(250×4.6 mm, Alltech, Deerfield, IL, USA) was used. The
mobile phase was composed of 50:50 V/V water and
acetonitrile at 1 mL/min. Lapatinib and paclitaxel concentration was calculated based on peak area.
MTT assay
Drug-resistant prostate cancer cell DU145-TXR was used
to determine the cell growth inhibition ability of drugloaded micelles. Cells were cultured in DMEM media
supplemented with 10% fetal bovine serum and 1%
antibiotic–antimycotic at 37°C in humidified environment
of 5% CO2. Cells were seeded in 96-well plates at a density
of 5,000 cells per well before treatment with drug-loaded
micelles as indicated. At the end of treatment, cell culture
medium was replaced by 100 μL medium with 0.5 mg/mL
3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and incubated for 1 h at 37°C. Then, the
medium was removed and 200 μL of DMSO was added
into each well to dissolve the formazan crystals. The
absorbance was measured in a microplate reader at a
wavelength of 560 nm. Cell viability was expressed as
Drug Deliv. and Transl. Res. (2011) 1:420–428
the percentage of control group. Cell viability (%)=
(Atest/Acontrol)×100%. DMSO controls were included in all
experiments where it was used to solubilize paclitaxel and
lapatinib.
Calcein AM assay
Cells were seeded into a black wall clear bottom 96-well
plate at a density of 20,000 cells per well a day before
experiment. After treating cells with various concentrations
of test compounds in 50 μL Dulbecco’s phosphate buffered
saline (DPBS) for 20 min at 37°C, 50 μL calcein AM
(10 μM) in DPBS were added to each well and incubated at
37°C for additional 20 min. Fluorescent intensity in each
well was determined using a SpectraMax M2/M2e spectrofluorometer (Sunnyvale, CA, USA) at the excitation
wavelength of 494 nm and emission wavelength of 517 nm.
Propidium iodide staining and cell cycle analysis
Cells were cultured in a 24-well plate to 90% confluence
and then treated for 24 h. Cells were trypsinized and fixed
in 70% ice-cold ethanol. After washing with PBS containing RNAse (1 mg/mL), cell pellet was re-suspended in
5 μg/mL propidium iodide staining solution for 15 min at
the room temperature. Cell cycle distribution was measured
by flow cytometry (Becton, Dickinson, NJ, USA). Results
from 10,000 fluorescent events were obtained for analysis.
TUNEL assay
TdT-mediated dUTP nick-end labeling (TUNEL) assay was
performed using DeadEnd Fluorometric TUNEL System
(Promega, Madison, WI, USA). Briefly, DU145-TXR cells
were grown on Lab-tek chamber slide and treated with
lapatinib, paclitaxel, and their combination for 24 h. At the
end of treatment, cells were fixed with acetone at −20°C for
10 min, washed with PBS, and equilibrated with buffer at
the room temperature for 10 min. Then, it was incubated
with a staining agent, composed of nucleotide and rTdT, in
a humidified chamber at 37°C for 1 h and protected from
light. The reaction was terminated with 2× standard sodium
citrate buffer and washed with PBS to remove unincorporated fluorescein-12-dUTP. The washed specimens were
then counterstained with 4′, 6-diamidino-2-phenylindole
(DAPI) and visualized with Zeiss AxioVision fluorescent
microscope.
In vivo anticancer efficacy study
All animal experiments were performed in accordance with
the NIH animal use guidelines and the protocol approved
by the Animal Care and Use Committee at the University of
Drug Deliv. and Transl. Res. (2011) 1:420–428
423
Tennessee Health Science Center. Xenograft flank tumors
were established in 8-week-old male athymic nude mice
(Charles River) by subcutaneous injection of five million
DU145-TXR prostate cancer cells suspended in 1:1 media
and matrigel. When tumors reached approximately
100 mm3, mice were randomized and assigned to different
treatments. PEG–PCD micelles containing both lapatinib
and paclitaxel were injected twice a week at the dose of
5 mg/kg each as a combination therapy via the tail vain
injection. For the monotherapy, micelles carrying either
lapatinib or paclitaxel were also injected at the dose of
10 mg/kg. Tumors were measured with a caliper prior to
each injection, and their volumes were calculated using the
formula: (width2 ×length)/2. At the end of study, tumor
tissue was excised from the mice and weighed.
Results
Effect of lapatinib and paclitaxel on MDR prostate cancer
cells
To determine whether lapatinib can reverse MDR in
prostate cancer cells, paclitaxel resistant prostate cancer
cells DU145-TXR were treated with the combination of
paclitaxel and lapatinib. As shown in Fig. 2, the IC50 of
paclitaxel was 2,069±597 nM in the absence of lapatinib,
indicating DU145-TXR cells were resistant to paclitaxel.
However, the IC50 of paclitaxel decreased to 79±29 nM at
lapatinib concentration of 1 μM and to 15±8 nM at
lapatinib concentration of 5 μM. These results suggest that
lapatinib can sensitize DU145-TXR to paclitaxel. This
study proved the feasibility of using lapatinib and paclitaxel
combination to overcome MDR in prostate cancers.
Viable Cells (%)
120
100
Preparation and characterization of drug-loaded
lipopolymer micelles
PEG–PCD lipopolymer were synthesized as described
previously [5]. The synthesized PEG–PCD lipopolymer
were characterized with 1H NMR, GPC. The Mn calculated
based on 1H NMR is 11,560. We also used GPC to
determine the apparent Mn and Mw of lipopolymer PEG–
PCD, which was 10,800 and 14,986, respectively.
To determine paclitaxel and lapatinib loading efficiency
into PEG–PCD lipopolymer micelles, drug concentration in
the formulations was determined by HPLC after dissolving
the drug-loaded micelles with acetonitrile. Due to the
different retention time for paclitaxel (4.7 min) and
lapatinib (7.1 min), we could simultaneously determine
their concentrations (Fig. 3a). We first tested the ability of
PEG–PCD micelles to load a single drug, either paclitaxel
or lapatinib. As shown in Fig. 3b, c, both these drugs could
be effectively loaded into lipopolymer micelles. For both
paclitaxel and lapatinib, the final drug concentration in the
formulation increased from around 0.5 to 2 mg/mL with
increase in theoretical loading (weight ratio of initial drug/
polymer) from 5% to 20%. The drug loading efficiency was
almost 100%, indicating all the drugs can be effectively
loaded into micelles at this range. As shown in Fig. 3d, we
could simultaneously load both paclitaxel and lapatinib into
PEG–PCD micelles and reach final drug concentration to at
least 1 mg for each drug. The drug loading efficiencies
were also around 100% for both drugs in all of these three
formulations. The particle size of PEG–PCD was approximately 60 nm for all the tested formulations, and the
incorporation of drugs had almost no effect on particle size
and the inhibition of MDR prostate cancer cell proliferation
by paclitaxel- and lapatinib-loaded lipopolymer micelles.
Effect of drug-loaded PEG–PCD micelles on DU145-TXR
cells
PTX
PTX+LAPA 1µM
80
PTX+LAPA 5µM
60
40
20
0
0
500
1000
1500
2000
PTX nM
PTX
PTX + 1µM LAPA
PTX+ 5µM LAPA
IC50 SD (nM)
2069 597
79 29
15 8
fold reversal
26.2
138.0
Fold of reversal = IC 50, no Lapatinib / IC50, with Lapatinib
Fig. 2 Effect of lapatinib on reversing MDR in DU145-TXR cells.
Percentage of viable cells was determined by MTT assay at 48 h
post-treatment. Data are the means±SD (n=4). Fold of reversal=
IC50, no lapatinib/IC50, with lapatinib. PTX paclitaxel, LAPA lapatinib
The anticancer effect of paclitaxel- and lapatinib-loaded
PEG–PCD micelles were tested in vitro on MDR
prostate cancer cells DU145-TXR (Fig. 4). No obvious
toxicity was observed in cells treated with blank micelles,
lapatinib (2 μM) or paclitaxel (0.5 μM) alone for 48 h,
while the combination of lapatinib (2 μM) and paclitaxel
(0.5 μM) significantly reduced the cell viability to around
23% of the control group. The combination of lapatinib
(1 μM) and paclitaxel (0.25 μM) also showed significant
inhibition of DU145-TXR cell proliferation, with the cell
viability of 35% of the control group. These results further
confirmed that lapatinib and paclitaxel-loaded PEG–PCD
micelles can overcome MDR in prostate cells and
effectively inhibit the proliferation of resistant prostate
cancer cells in vitro.
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Drug Deliv. and Transl. Res. (2011) 1:420–428
A
B
2
Concentration
(mg/mL)
0.014
0.012
0.010
AU
Fig. 3 Determination of drug
concentrations in paclitaxeland/or lapatinib-loaded
lipopolymer micelles. a HPLC
chromatography of paclitaxel
and lapatinib; b effect of
theoretical loading on paclitaxel
solubility; c effect of theoretical
loading on lapatinib solubility; d
drug concentration in paclitaxel
and lapatinib co-loaded
lipopolymer micelles
0.008
Lapatinib
0.006
0.004
0.002
Paclitaxel
Paclitaxel
1.5
1
0.5
0
5
10
20
Theoretical Loading (%)
0.000
1
2
3
4
5
6
7
8
9
Retention time (Min)
D
Drug Concentration
(mg/mL)
C
Concentration
( mg/mL)
2.5
2
Lapatinib
1.5
1
0.5
0
5
10
20
Theoretical Loading (%)
Inhibition of P-gp activity in MDR cancer cells by lapatinib
To determine whether lapatinib overcomes MDR in
cancer cells, we performed calcein AM assay to
determine the effect of lapatinib on P-gp activity [6].
The intracellular accumulation of calcein, which is a
fluorescent P-gp substrate, was observed with a fluorescent microscope and also quantitatively determined by a
spectrofluorometer. As shown in Fig. 5a, lapatinib treatment led to dose-dependent increase in intracellular
calcein fluorescence. The fluorescent intensity increased
with increase in lapatinib concentration from 0 to 20 μM.
120
2.5
Paclitaxel
Lapatinib
2
1.5
1
0.5
0
Polymer (mg)
Paclitaxel (mg)
Lapatinib (mg)
10
0.5
0.5
20
1
1
10
1
1
We also quantitatively determined the fluorescent intensity
with a spectrofluorometer (Fig. 5b). In the absence of
lapatinib, the fluorescence intensity was approximately
50 RFU; however, it increased to around 800 RFU at a
lapatinib concentration of 20 μM. In contrast, treatment
with blank PEG–PCD micelles showed negligible effect
on intracellular accumulation of calcein fluorescence.
We next examined the effect of lapatinib on P-gp
transport at the molecular level. From Fig. 5c, d, parent
DU145 cells did not express MDR-1 and P-gp at
detectable levels. In contrast, P-gp was overexpressed in
DU145-TXR cells. Also, the treatment of DU145-TXR
cells with 2 μM lapatinib for 24 h did not alter P-gp
expression at both mRNA (Fig. 5c) and protein levels
(Fig. 5d).
Viable Cells (%)
100
Cell cycle and apoptosis of MDR prostate cancer cells
80
60
40
*
20
*
0
NT
PEG-PCD LAPA 2µM PTX 0.5µM LAPA 2 µM LAPA 1µM
PTX 0.5µM PTX 0.25µM
Fig. 4 Anticancer effect of paclitaxel and lapatinib combination on
MDR DU145-TXR cells. DU145-TXR cells were treated with
micelles containing either lapatinib alone, paclitaxel alone, or
combination of these two drugs for 48 h, and then cell viability was
determined by MTT assay and expressed as a percentage of control.
*p<0.01, compare with non-treated control (NT). PTX paclitaxel,
LAPA lapatinib
We also determined the effect of lapatinib- and paclitaxelloaded PEG–PCD micelles on DU145-TXR cells by
analyzing cell cycle distribution and cell apoptosis by flow
cytometry. Cells were treated with different formulations
for 24 h before analysis. As shown in Fig. 6, the treatment
of lapatinib (2 μM) or paclitaxel (0.5 μM) alone had no
effects on the cycle distribution. In contrast, their combination showed significant effects on cycle distribution and
cell apoptosis. The treatment of 2 μM lapatinib+0.5 μM
paclitaxel caused cells in G0/G1 phase decreased from
29.0±2.1% in the control group to 4.7±0.2% in the
Drug Deliv. and Transl. Res. (2011) 1:420–428
425
B
0
1
5
Lapatinib (µM)
Fluorescence Intensity
A
20
Lapatinib (µM)
1000
800
600
400
200
0
0
5
10
15
20
Lapatinib (µM)
C
120
Control
DU145
MDR -1 mRNA level
(% of control)
DU145-TXR
LAPA
MDR1
S19
100
80
60
40
20
0
Control
LAPA
DU145
DU145-TXR
D
DU145
DU145-TXR
Control
LAPA
P-gp
GAPDH
Fig. 5 Effect of lapatinib on P-gp activity inhibition and expression. a
Fluorescent images depicting dose-dependent increase in intracellular
calcein fluorescence in DU145-TXR following treatment with
lapatinib (2 μM). b Intracellular calcein accumulation in DU145 or
DU145-TXR cells. c P-gp mRNA expression following treatment with
2 μM lapatinib for 24 h. d P-gp protein expression after 24 h treatment
with 2 μM lapatinib. PTX paclitaxel, LAPA lapatinib
combination group. Meanwhile, cells in G2/M phase
increased from 50.7±4.0% in the control group to 61.1±
1.2% in the combination group. In addition, the apoptotic
sub G1 phase cells increased from 0.4±0.2% in the control
group to 25.1±2.5% in the combination group. These
results indicated that the cells treated with lapatinib and
paclitaxel combination therapy failed to undergo mitosis.
This is consistent with the anticancer mechanism of
A
B
G0/G1
G2/M
S
SubG1
70
**
60
LAPA+PTX
PTX
LAPA
Control
% Cells
50
40
*
30
*
20
10
*
0
Fig. 6 Effect of paclitaxel and lapatinib combination on cell cycle and
apoptosis of MDR prostate cancers. DU145-TXR cells were treated
with PEG–PCD micelles formulated lapatinib (2 μM), paclitaxel
(0.5 μM), and lapatinib (2 μM)+paclitaxel (0.5 μM). After 24 h
Control
LAPA
PTX
LAPA+PTX
treatment, cell cycles were analyzed by flow cytometry after PI
staining. a Cell cycle distribution; b quantitative analysis. *p<0.01;
**p<0.05, compare with control. PTX paclitaxel, LAPA lapatinib
426
Drug Deliv. and Transl. Res. (2011) 1:420–428
paclitaxel, which is a microtubule inhibitor and can cause the
arrest of the cell cycle at mitotic phase. The increased cell
population in sub-G1 phase indicated that apoptotic cells were
increased after treating with the combination therapy [25].
We also used TUNEL assay to further confirm that the
combination of paclitaxel and lapatinib induced apoptosis
in DU145-TXR cells. As shown in Fig. 7, neither paclitaxel
(0.5 μM) nor lapatinib (2 μM) alone caused apoptotic cell
death in DU145-TXR cells, while the combination of
paclitaxel (0.5 μM) and lapatinib (2 μM) induced significant cell apoptosis.
Effect of lapatinib and paclitaxel combination on tumor
microvasculature
Lapatinib- and paclitaxel-loaded lipopolymer micelles
overcome MDR in vivo
Discussion
Encouraged by the in vitro anticancer effect of lapatiniband paclitaxel-loaded PEG–PCD micelles in MDR prostate
cancer cells, we tested their ability to overcome MDR in
vivo using athymic nude mouse xenograft model established with DU145-TXT MDR prostate cancer cells. When
tumors reached approximately 100 mm3, mice were
randomized into four groups of eight mice. Mice were
treated with the tail vain injection of PEG–PCD micelles at
a dose of 10 mg/kg paclitaxel as the monotherapy group or
5 mg/kg paclitaxel + 5 mg/kg lapatinib in the combination
therapy group, respectively. In contrast to paclitaxel
monotherapy, lapatinib and paclitaxel combination therapy
significantly inhibited in vivo tumor growth (Fig. 8a). We
also determined the weight of tumor at the end of the study,
which also showed that the weight of tumors in the
combination group was significantly less than those in
paclitaxel monotherapy group. In addition, we did not
observe any mortality and significant decrease body weight
in mice after drug administration, indicating the negligible
toxicity of PEG–PCD lipopolymer micelles used in this
study (data not shown).
A
We examined the vessel density of xenograft tumors to
determine the effect of lapatinib and paclitaxel combination
on tumor neovascularization (Fig. 8b). While lapatinib
treatment had some effect on angiogenesis, the combination
of these two drugs strongly inhibited angiogenesis (less
CD31-positive staining) compared to paclitaxel monotherapy
or control.
Hormone refractory prostate cancer is aggressive and resistant
to prolonged treatment with paclitaxel and other chemotherapeutic agents due to the activity of cell surface drug transporters responsible for multidrug resistance. P-glycoprotein is
one commonly overexpressed ABC transporters in a variety of
MDR cancers including prostate cancer [26]. A promising
approach to treat cancers overexpressing P-gp is to combine
traditional chemotherapy drugs with P-gp inhibitors.
Recently, various tyrosine kinase inhibitors including
lapatinib have been shown to interact with and/or inhibit
the activity of P-gp. Consequently, we hypothesize that
co-administration of lapatinib and paclitaxel may potently kill MDR cancers since lapatinib will sensitize
cells and potentiate paclitaxel activity.
Our findings confirmed that lapatinib works by inhibiting P-gp function but not decreasing transporter expression
since we observed dose-dependent increase in intracellular
calcein fluorescence in DU145-TXR following treatment
with lapatinib, but no effect on MDR-1 mRNA and P-gp
protein levels. These results closely agree with the work of
Dai et al. who showed similar results for MCF-7/adr cells
[12]. Furthermore, Dunne and coworkers observed no
B
C
TUNEL/DAPI
Paclitaxel 0.5 µM
TUNEL/DAPI
Lapatinib 2 µM
Fig. 7 Detection of paclitaxel and lapatinib combination induced cell
apoptosis with TUNEL staining. Paclitaxel and lapatinib combination
induced cell apoptosis in DU145-TXR Cells. DU145 cells were
treated with a paclitaxel 0.5 μM, b lapatinib 2 μM, and c paclitaxel
TUNEL/DAPI
Paclitaxel 0.5 µM
+Lapatinib 2 µM
0.5 μM+lapatinib 2 μM. After 24 h treatment, cells were subject to
TUNEL staining and counterstained with DAPI. Images are overlay of
green fluorescence from the TUNEL stain with blue fluorescence from
DAPI
Drug Deliv. and Transl. Res. (2011) 1:420–428
A
1500
427
B
Control
Tumor Volume (mm3)
LAPA 10mg/Kg
PTX 10mg/Kg
1000
LAPA 5mg/Kg + PTX 5mg/Kg
500
*
*
*
0
0
Injection
5
*
*
10
15
20
Days
Fig. 8 In vivo anticancer effect of paclitaxel- and lapatinib-loaded
lipopolymer micelles on xenograft MDR prostate tumors. a Nude
mice bearing 100-mm3 DU145-TXR tumors were given intravenous
injection of PEG–PCD micelles containing lapatinib and paclitaxel
were injected twice a week at a dose of 10 mg/kg for monotherapy
and 5 mg/kg per drug for combination therapy. Tumor growth
regression was significantly higher for combination therapy compared
to monotherapy and control. Points are mean tumor size (n=8); bars,
SE. b Tumor vessels for each treatment were stained with anti-CD31
antibody to assess antiangiogenic activity. PTX paclitaxel, LAPA
lapatinib
change in MDR1 mRNA levels upon treating A549-tax
cells with increasing concentrations of lapatinib [26].
However, in contrast with our results (Fig. 5c, d), they
observed a dose-dependent increase in P-gp protein levels
which was attributed to posttranscriptional effects. Since we
used similar doses of lapatinib in our study as Dunne et al., it is
not clear what the underlying mechanistic differences are or
whether these differences are merely cell line dependent.
We developed paclitaxel- and lapatinib-loaded lipopolymer
micelle formulations with high drug loading efficiency, small
particle size (~60 nm) and narrow size distribution and
performed in vitro cell-based studies to test our hypothesis.
Paclitaxel (0.5 μM) and lapatinib (2 μM) combination
effectively inhibited DU145-TXR cell proliferation in vitro,
induced cell cycle perturbation, and increased cell apoptosis.
In contrast, monotherapy with 0.5 μM paclitaxel or 2 μM
lapatinib alone showed almost no effect on DU145-TXR cells.
Co-administration of lapatinib and paclitaxel resulted in a 26and 138-fold reversal when lapatinib concentration increased
from 1 to 5 μM. It is also noteworthy that decreasing the
concentration of both drugs by 50% (i.e., 0.25 μM paclitaxel
and 1 μM lapatinib) still resulted in a decrease in cell viability
to approximately 30%. Our findings are supported by several
in vitro data published in the literature. Coley et al. [27] have
shown that the combination of GW282974A (lapatinib
analog) and paclitaxel is synergistic in inhibiting proliferation of resistant ovarian cancer cell line (PEO1TaxR) [27].
Lapatinib has also been shown to increase cytotoxic activity
of SN-38 and Topotecan in resistant glioblastoma, colorectal
carcinoma, and ABCG2-transfected breast cancer cells [28].
Similar to in vitro study, the combination of 5 mg/kg
paclitaxel and 5 mg/kg lapatinib significantly inhibited
tumor growth in vivo compared with 10 mg/kg paclitaxel
monotherapy (Fig. 8). These studies indicate that paclitaxeland lapatinib-loaded PEG–PCD lipopolymer micelles could
be used to treat MDR prostate cancers. Since tumor growth
requires formation of new blood vessels from pre-existing
vessels, we hypothesized that combination of lapatinib and
paclitaxel will be more potent compared to monotherapy in
inhibiting angiogenic activity. To assess tumor vessel
density, we performed immunohistochemical staining with
anti-CD31 antibody. Our results suggest paclitaxel alone
did not significantly alter microvessel density compared to
control. In contrast, combined treatment with these two
drugs appeared to be synergistic in decreasing antiangiogenic activity compared to the control or monotherapy.
Although paclitaxel has been shown to exhibit antiangiogenic activity [29], our observations were different. This
may possibly be due to the lower dose (10 vs 28 mg/kg)
and paclitaxel resistant cells used in our study. Lapatinib is
a selective and potent dual tyrosine kinase inhibitor of
EGFR and HER2 and can stabilize tumor progression in
monotherapy, a fact confirmed by our results in Fig. 8b. It
also acts synergistically in combination with chemotherapy
[30]. The antineoplastic activity of lapatinib and paclitaxel
combination seen in our study suggests that at the
concentrations used for the study, both inhibitions of
angiogenesis as well as direct cytotoxic effects may be
occurring. Further experiments to clarify the antineoplastic
mechanisms and dysregulated signaling pathways involved
in prostate tumors need to be carried out.
Taken together our results confirm that the lipopolymer
PEG–PCD successfully delivered a combination of lapatinib and paclitaxel to prostate cancer cells in vitro and in
428
vivo. Lapatinib reversed MDR in drug-resistant tumors and
paclitaxel enhanced toxicity in P-gp overexpressing prostate cancer cells. Finally, our non-toxic lipopolymer
described here avoids the use of solubilizing agents often
required for formulating hydrophobic anticancer drugs,
thereby avoiding any potential systemic or organ toxicity.
Acknowledgments This work is supported by an Idea Award
(W81XWH-10-1-0969) from the Department of Defense Prostate
Cancer Research Program.
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